Hysteresis in the Thermo-Responsive Assembly of Hexa(ethylene glycol) Derivative-Modified Gold Nanodiscs as an Effect of Shape.

Anisotropic gold nanodiscs (AuNDs) possess unique properties, such as large flat surfaces and dipolar plasmon modes, which are ideal constituents for the fabrication of plasmonic assemblies for novel and emergent functions. In this report, we present the thermo-responsive assembly and thermo-dynamic behavior of AuNDs functionalized with methyl-hexa(ethylene glycol) undecane-thiol as a thermo-responsive ligand. Upon heating, the temperature stimulus caused a blue shift of the plasmon peak to form a face-to-face assembly of AuNDs due to the strong hydrophobic and van der Waals interactions between their large flat surfaces. Importantly, AuNDs allowed for the incorporation of the carboxylic acid-terminated ligand while maintaining their thermo-responsive assembly ability. With regard to their reversible assembly/disassembly behavior in the thermal cycling process, significant rate-independent hysteresis, which is related to their thermo-dynamics, was observed and was shown to be dependent on the carboxylic acid content of the surface ligands. As AuNDs have not only unique plasmonic properties but also high potential for attachment due to the fact of their flat surfaces, this study paves the way for the exploitation of AuNDs in the development of novel functional materials with a wide range of applications.

Probing the Biogenesis of Polysaccharide Granules in Algal Cells at Sub-Organellar Resolution via Raman Microscopy with Stable Isotope Labeling.

Phototrophs assimilate CO2 into organic compounds that accumulate in storage organelles. Elucidation of the carbon dynamics of storage organelles could enhance the production efficiency of valuable compounds and facilitate the screening of strains with high photosynthetic activity. To comprehensively elucidate the carbon dynamics of these organelles, the intraorganellar distribution of the carbon atoms that accumulate at specific time periods should be probed. In this study, the biosynthesis of polysaccharides in storage organelles was spatiotemporally probed via stimulated Raman scattering (SRS) microscopy using a stable isotope (13C) as the tracking probe. Paramylon granules (a storage organelle of ß-1,3-glucan) accumulated in a unicellular photosynthetic alga, Euglena gracilis, were investigated as a model organelle. The carbon source of the culture medium was switched from NaH12CO3 to NaH13CO3 during the production of the paramylon granules; this resulted in the distribution of the 12C and 13C constituents in the granules, so that the biosynthetic process could be tracked. Taking advantage of high-resolution SRS imaging and label switching, the localization of the 12C and 13C constituents inside a single paramylon granule could be visualized in three dimensions, thus revealing the growth process of paramylon granules. We propose that this method can be used for comprehensive elucidation of the dynamic activities of storage organelles.

Molecular configuration-mediated thermo-responsiveness in oligo(ethylene glycol) derivatives attached on gold nanoparticles.

Biomolecular systems actively control their local environment on a sub-nm scale via changes in molecular configuration from their flexible structures and derive emergent functions. Although this functional emergence based on local environmental control is attracting a great deal of attention in chemistry, it remains challenging to realize this artificially. Herein, we report the tuning of the thermo-responsive properties of oligo(ethylene glycol) (OEG) derivatives attached on gold nanoparticles via local environmental control not only by the hydrophobic moiety at their terminus but also by their molecular configuration. OEG-attached alkane thiol-modified AuNPs showed thermo-responsive assembly/disassembly in water through the hydration/dehydration of the OEG portions in a manner dependent both on the hydrophobicity at their terminus and the surface curvature of the core nanoparticles. Further, the assembly temperature (T A) was also tuned by ligand mixing with a non-thermo-responsive ligand with a shorter OEG length. Molecular dynamics simulations show that the distribution of the hydrophobic terminus in the normal direction along the gold surface varied in accordance with the surface curvature, indicating variations in molecular configuration. It is expected that a bent configuration could accelerate the thermo-responsiveness of OEG by allowing them greater accessibility to the hydrophobic terminus. Experimental and simulation results support the notion that local OEG density tuning by surface curvature or ligand mixing with a different OEG length leads to different degrees of accessibility to the hydrophobic terminus via changes in molecular configuration, promoting local environmental control-directed assembly temperature tuning.

Raman image-activated cell sorting.

The advent of image-activated cell sorting and imaging-based cell picking has advanced our knowledge and exploitation of biological systems in the last decade. Unfortunately, they generally rely on fluorescent labeling for cellular phenotyping, an indirect measure of the molecular landscape in the cell, which has critical limitations. Here we demonstrate Raman image-activated cell sorting by directly probing chemically specific intracellular molecular vibrations via ultrafast multicolor stimulated Raman scattering (SRS) microscopy for cellular phenotyping. Specifically, the technology enables real-time SRS-image-based sorting of single live cells with a throughput of up to ~100 events per second without the need for fluorescent labeling. To show the broad utility of the technology, we show its applicability to diverse cell types and sizes. The technology is highly versatile and holds promise for numerous applications that are previously difficult or undesirable with fluorescence-based technologies.

Spatiotemporal monitoring of intracellular metabolic dynamics by resonance Raman microscopy with isotope labeling.

Cellular metabolites are valuable in a diverse range of applications. For example, the unicellular green alga Haematococcus lacustris produces as a secondary metabolite the carotenoid pigment astaxanthin (AXT), which is widely used in nutraceutical, cosmetic, and food industries due to its strong antioxidant activity. In order to enhance the productivity of H. lacustris, spatial and temporal understanding of its metabolic dynamics is essential. Here we show spatiotemporal monitoring of AXT production in H. lacustris cells by resonance Raman microscopy combined with stable isotope labeling. Specifically, we incorporated carbon dioxide (13CO2) labeled with a stable isotope (13C) into H. lacustris cells through carbon fixation and traced its conversion to 13C-AXT using our resonance Raman microscope. We incubated H. lacustris cells under various conditions by switching, pulsing, and replacing 13CO2 and 12CO2. By measurement of these cells we determined the fixation time of 13C-carbon, visualized the intracellular localization of 13C- and 12C-AXTs, and revealed the dynamic consumption-production equilibrium of the accumulated AXT. This work is a valuable step in the development of effective screening criteria for high AXT-producing H. lacustris cells.

Reversible changes in the orientation of gold nanorod arrays on polymer brushes.

Nanoparticles exhibit a number of unique properties such as localized surface plasmon resonance (LSPR). As this LSPR is sensitive to geometrical or spatial conditions, the arrangement of nanoparticles, in particular the active arrangement of plasmonic structures, is an important issue. In this study, gold nanorod (GNR) arrays were prepared by GNR attachment on anionic polymer (DNA) brushes via electrostatic interactions and their stimuli-responsive changes in orientation were investigated. As a result, the orientation of GNR arrays on DNA brushes reversibly changed by the modulation of electrostatic interactions between GNRs and polymers via changes in the solution pH. As these extensive GNR arrays are prepared via easy bottom-up processes, GNR surface properties are easily tuned by simple modification, and DNAs could be replaced with various synthetic polymers, we believe that this study will lead to the development of next-generation materials and devices with actively tunable structures.

Homogeneous Oligomeric Ligands Prepared via Radical Polymerization that Recognize and Neutralize a Target Peptide.

Abiotic ligands that bind to specific biomolecules have attracted attention as substitutes for biomolecular ligands, such as antibodies and aptamers. Radical polymerization enables the production of robust polymeric ligands from inexpensive functional monomers. However, little has been reported about the production of monodispersed polymeric ligands. Herein, we present homogeneous ligands prepared via radical polymerization that recognize epitope sequences on a target peptide and neutralize the toxicity of the peptide. Taking advantage of controlled radical polymerization and separation, a library of multifunctional oligomers with discrete numbers of functional groups was prepared. Affinity screening revealed that the sequence specificity of the oligomer ligands strongly depended on the number of functional groups. The process reported here will become a general step for the development of abiotic ligands that recognize specific peptide sequences.

Isolating Single Euglena gracilis Cells by Glass Microfluidics for Raman Analysis of Paramylon Biogenesis.

Time-course analysis of single cells is important to characterize heterogeneous activities of individual cells such as the metabolic response to their environment. Single-cell isolation is an essential step prior to time-course analysis of individual cells by collecting, culturing, and identifying multiple single-cell targets. Although single-cell isolation has been performed by various methods previously, a glass microfluidic device with semiclosed microchannels dramatically improved this process with its simple operation and easy transfer for time-course analysis of identified single cells. This study demonstrates isolating single cells of the highly motile microalgae, Euglena gracilis, by semiclosed microchannels with liquid flow only. The isolated single cells were identified in isolating channels and continuously cultured to track, by Raman microscopy, for the formation of subcellular granules composed of polysaccharide paramylon, a unique metabolite of E. gracilis, generated through photosynthesis. Through low-temperature glass bonding, a thin glass interface was incorporated to the microfluidic device. Thus, the device could perform the direct measurements of cultured single cells at high magnification by Raman microscopy with low background noise. In this study, the first demonstration of sequential monitoring of paramylon biogenesis in a single identified E. gracilis cell is shown.

High-throughput label-free molecular fingerprinting flow cytometry.

Flow cytometry is an indispensable tool in biology for counting and analyzing single cells in large heterogeneous populations. However, it predominantly relies on fluorescent labeling to differentiate cells and, hence, comes with several fundamental drawbacks. Here, we present a high-throughput Raman flow cytometer on a microfluidic chip that chemically probes single live cells in a label-free manner. It is based on a rapid-scan Fourier-transform coherent anti-Stokes Raman scattering spectrometer as an optical interrogator, enabling us to obtain the broadband molecular vibrational spectrum of every single cell in the fingerprint region (400 to 1600 cm-1) with a record-high throughput of ~2000 events/s. As a practical application of the method not feasible with conventional flow cytometry, we demonstrate high-throughput label-free single-cell analysis of the astaxanthin productivity and photosynthetic dynamics of Haematococcus lacustris.

Conformation Manipulation and Motion of a Double Paddle Molecule on an Au(111) Surface.

The molecular conformation of a bisbinaphthyldurene (BBD) molecule is manipulated using a low-temperature ultrahigh-vacuum scanning tunneling microscope (LT-UHV STM) on an Au(111) surface. BBD has two binaphthyl groups at both ends connected to a central durene leading to anti/syn/flat conformers. In solution, dynamic nuclear magnetic resonance indicated the fast interexchange between the anti and syn conformers as confirmed by density functional theory calculations. After deposition in a submonolayer on an Au(111) surface, only the syn conformers were observed forming small islands of self-assembled syn dimers. The syn dimers can be separated into syn monomers by STM molecular manipulations. A flat conformer can also be prepared by using a peculiar mechanical unfolding of a syn monomer by STM manipulations. The experimental STM dI/dV and theoretical elastic scattering quantum chemistry maps of the low-lying tunneling resonances confirmed the flat conformer BBD molecule STM production. The key BBD electronic states for a step-by-step STM inelastic excitation lateral motion on the Au(111) are presented requiring no mechanical interactions between the STM tip apex and the BBD. On the BBD molecular board, selected STM tip apex positions for this inelastic tunneling excitation enable the flat BBD to move controllably on Au(111) by a step of 0.29 nm per bias voltage ramp.

Monitoring Photosynthetic Activity in Microalgal Cells by Raman Spectroscopy with Deuterium Oxide as a Tracking Probe.

Microalgae offer great potential for the production of biofuel, but high photosynthetic activity is demanded for the practical realisation of microalgal biofuels. To this end, it is essential to evaluate the photosynthetic activity of single microalgal cells in a heterogeneous population. In this study, we present a method to monitor the photosynthetic activity of microalgae (in particular Euglena gracilis, a microalgal species of unicellular, photosynthetic, flagellate protists as our model organism) at single-cell resolution by Raman spectroscopy with deuterium from deuterium oxide (D2 O) as a tracking probe. Specifically, we replaced H2 O in culture media with D2 O up to a concentration of 20 % without disturbing the growth rate of E. gracilis cells and evaluated C-D bond formation as a consequence of photosynthetic reactions by Raman spectroscopy. We used the probe to monitor the kinetics of the C-D bond formation in E. gracilis cells by incubating them in D2 O media under light irradiation. Furthermore, we demonstrated Raman microscopy imaging of each single E. gracilis cell to discriminate deuterated cells from normal cells. Our results hold great promise for Raman-based screening of E. gracilis and potentially other microalgae with high photosynthetic activity by using D2 O as a tracking probe.

A polymer nanoparticle with engineered affinity for a vascular endothelial growth factor (VEGF165).

Protein affinity reagents are widely used in basic research, diagnostics and separations and for clinical applications, the most common of which are antibodies. However, they often suffer from high cost, and difficulties in their development, production and storage. Here we show that a synthetic polymer nanoparticle (NP) can be engineered to have many of the functions of a protein affinity reagent. Polymer NPs with nM affinity to a key vascular endothelial growth factor (VEGF165) inhibit binding of the signalling protein to its receptor VEGFR-2, preventing receptor phosphorylation and downstream VEGF165-dependent endothelial cell migration and invasion into the extracellular matrix. In addition, the NPs inhibit VEGF-mediated new blood vessel formation in Matrigel plugs in vivo. Importantly, the non-toxic NPs were not found to exhibit off-target activity. These results support the assertion that synthetic polymers offer a new paradigm in the search for abiotic protein affinity reagents by providing many of the functions of their protein counterparts.

Mechanically Induced Opening-Closing Action of Binaphthyl Molecular Pliers: Digital Phase Transition versus Continuous Conformational Change.

Reversible dynamic control of structure is a significant challenge in molecular nanotechnology. Previously, we have reported a mechanically induced continuous (analog) conformational variation in an amphiphilic binaphthyl, where closing of molecular pliers was achieved by compression of a molecular monolayer composed of these molecules at the air-water interface. In this work we report that a phase transition induced by an applied mechanical stress enables discontinuous digital (1/0) opening of simple binaphthyl molecular pliers. A lipid matrix at the air-water interface promotes the formation of quasi-stable nanocrystals, in which binaphthyl molecules have an open transoid configuration. The crystallization/dissolution of quasi-stable binaphthyl crystals with accompanying conformational change is reversible and repeatable.

Supramolecular 1-D polymerization of DNA origami through a dynamic process at the 2-dimensionally confined air-water interface.

In this study, a Langmuir-Blodgett (LB) system has been utilized for the regulation of polymerization of a DNA origami structure at the air-water interface as a two-dimensionally confined medium, which enables dynamic condensation of DNA origami units through variation of the film area at the macroscopic level (ca. 10-100 cm(2)). DNA origami sheets were conjugated with a cationic lipid (dioctadecyldimethylammonium bromide, 2C18N(+)) by electrostatic interaction and the corresponding LB-film was prepared. By applying dynamic pressure variation through compression-expansion processes, the lipid-modified DNA origami sheets underwent anisotropic polymerization forming a one-dimensionally assembled belt-shaped structure of a high aspect ratio although the thickness of the polymerized DNA origami was maintained at the unimolecular level. This approach opens up a new field of mechanical induction of the self-assembly of DNA origami structures.

In situ 2D-extraction of DNA wheels by 3D through-solution transport.

Controlled transfer of DNA nanowheels from a hydrophilic to a hydrophobic surface was achieved by complexation of the nanowheels with a cationic lipid (2C12N(+)). 2D surface-assisted extraction, '2D-extraction', enabled structure-persistent transfer of DNA wheels, which could not be achieved by simple drop-casting.

Mechanochemical Tuning of the Binaphthyl Conformation at the Air-Water Interface.

Gradual and reversible tuning of the torsion angle of an amphiphilic chiral binaphthyl, from -90° to -80°, was achieved by application of a mechanical force to its molecular monolayer at the air-water interface. This 2D interface was an ideal location for mechanochemistry for molecular tuning and its experimental and theoretical analysis, since this lowered dimension enables high orientation of molecules and large variation in the area. A small mechanical energy (<1 kcal mol(-1) ) was applied to the monolayer, causing a large variation (>50 %) in the area of the monolayer and modification of binaphthyl conformation. Single-molecule simulations revealed that mechanical energy was converted proportionally to torsional energy. Molecular dynamics simulations of the monolayer indicated that the global average torsion angle of a monolayer was gradually shifted.

Nanoarchitectonics of molecular aggregates: science and technology.

The field of making, studying and using molecular aggregates, in which the individual molecules (monomers) are arranged in a regular fashion, has come a long way. Taking control over the aggregation of small molecules and polymers in bulk, on surfaces and at interfaces pose a considerable challenge for their utilization in modern high tech applications. In this review, we provide a detailed insight into recent trends in molecular aggregates from the perspectives of nanoarchitectonics.

Epitope discovery for a synthetic polymer nanoparticle: a new strategy for developing a peptide tag.

We describe a novel epitope discovery strategy for creating an affinity agent/peptide tag pair. A synthetic polymer nanoparticle (NP) was used as the "bait" to catch an affinity peptide tag. Biotinylated peptide tag candidates of varied sequence and length were attached to an avidin platform and screened for affinity against the polymer NP. NP affinity for the avidin/peptide tag complexes was used to provide insight into factors that contribute NP/tag binding. The identified epitope sequence with an optimized length (tMel-tag) was fused to two recombinant proteins. The tagged proteins exhibited higher NP affinity than proteins without tags. The results establish that a fusion peptide tag consisting of optimized 15 amino acid residues can provide strong affinity to an abiotic polymer NP. The affinity and selectivity of NP/tMel-tag interactions were exploited for protein purification in conjunction with immobilized metal ion/His6-tag interactions to prepare highly purified recombinant proteins. This strategy makes available inexpensive, abiotic synthetic polymers as affinity agents for peptide tags and provides alternatives for important applications where more costly affinity agents are used.

Polymer nanoparticle-protein interface. Evaluation of the contribution of positively charged functional groups to protein affinity.

Cationic-functionalized polymer nanoparticles (NPs) show strikingly distinct affinities to proteins depending on the nature of the cationic functional group. N-Isopropylacrylamide (NIPAm) polymer NPs incorporating three types of positively charged functional groups (guanidinium, primary amino, and quaternary ammonium groups) were prepared by precipitation polymerization. The affinities to fibrinogen, a protein with an isoelectric point (pI) of 5.5, were compared using UV-vis spectrometry and a quartz crystal microbalance (QCM). Guanidinium-containing NPs showed the highest affinity to fibrinogen. The observation is attributed to strong, specific interactions with carboxylate groups on the protein surface. The affinity of the positively charged NPs to proteins with a range of pIs revealed that protein-NP affinity is due to a combination of ionic, hydrogen bonding, and hydrophobic interactions. Protein affinity can be modulated by varying the composition of these functional monomers in the acrylamide NPs. Engineered NPs containing the guanidinium group with hydrophobic and hydrogen bonding functional groups were used in an affinity precipitation for the selective separation of fibrinogen from a plasma protein mixture. Circular dichroism (CD) revealed that the protein was not denatured in the process of binding or release.

ELISA-mimic screen for synthetic polymer nanoparticles with high affinity to target proteins.

Synthetic polymer nanoparticles (NPs) that display high affinity to protein targets have significant potential for medical and biotechnological applications as protein capture agents or functional replacements of antibodies ("plastic antibodies"). In this study, we modified an immunological assay (enzyme-linked immunosorbent assay: ELISA) into a high-throughput screening method to select nanoparticles with high affinity to target proteins. Histone and fibrinogen were chosen as target proteins to demonstrate this concept. The selection process utilized a biotinylated NP library constructed with combinations of functional monomers. The screen identified NPs with distinctive functional group compositions that exhibited high affinity to either histone or fibrinogen. The variation of protein affinity with changes in the nature and amount of functional groups in the NP provided chemical insight into the principle determinants of protein-NP binding. The NP affinity was semiquantified using the ELISA-mimic assay by varying the NP concentrations. The screening results were found to correlate with solution-based assay results. This screening system utilizing a biotinylated NP is a general approach to optimize functional monomer compositions and can be used to rapidly search for synthetic polymers with high (or low) affinity for target biological macromolecules.